A key application of deep sequencing is gene expression analysis at the RNA level, commonly known as RNA-seq. Reads from RNA-seq cover all regions of an mRNA sequence. However, sequences derived from the 3' end region are sufficient to indicate gene expression levels. All 3' end sequencing methods have revealed that most eukaryotic genes display alternative cleavage and polyadenylation (APA), where RNA isoforms using different cleavage and polyadenylation sites (pAs) can be generated from a gene. Importantly, different APA isoforms have been shown to have different metabolisms, including stability, localization and translation. APA clearly poses a challenge for gene expression analysis. Current RNA-seq methods cannot capture this information; second, false pA identification due to internal priming leads to inaccurate APA information; third, complete analysis of gene expression needs to take into consideration all APA isoforms. We have proposed a new method that completely addresses false priming, incomplete 3' end annotation and significantly propels research on single cell RNA expression and poly(A) tail length, which have so far been elusive. The final goal of this proposal is to develop a commercial kit, allowing non-specialized labs the ability to examine 3'ends in an accurate and sensitive manner.